Expression Of OCN Research Articles

Comprehensive analysis of M2 macrophage-derived exosomes facilitating osteogenic differentiation of human periodontal ligament stem cells

BackgroundThe role of periodontal ligament stem cells (PDLSCs) and macrophage polarization in periodontal tissue regeneration and bone remodeling during orthodontic tooth movement (OTM) has been well documented. Nevertheless, the interactions between macrophages and PDLSCs in OTM remain to be investigated. Consequently, the present study was proposed to explore the effect of different polarization states of macrophages on the osteogenic differentiation of PDLSCs.MethodsAfter M0, M1 and M2 macrophage-derived exosomes (M0-exo, M1-exo and M2-exo) treatment of primary cultured human PDLSCs, respectively, mineralized nodules were observed by Alizarin red S staining, and the expression of ALP and OCN mRNA and protein were detected by RT-qPCR and Western blotting, correspondingly. Identification of differentially expressed microRNAs (DE-miRNA) in M0-exo and M2-exo by miRNA microarray, and GO and KEGG enrichment analysis of DE-miRNA targets, and construction of protein–protein interaction networks.ResultsM2-exo augmented mineralized nodule formation and upregulated ALP and OCN expression in PDLSCs, while M0-exo had no significant effect. Compared to M0-exo, a total of 52 DE-miRNAs were identified in M2-exo. The expression of hsa-miR-6507-3p, hsa-miR-4731-3p, hsa-miR-4728-3p, hsa-miR-3614-5p and hsa-miR-6785-3p was significantly down-regulated, and the expression of hsa-miR-6085, hsa-miR-4800-5p, hsa-miR-4778-5p, hsa-miR-6780b-5p and hsa-miR-1227-5p was significantly up-regulated in M2-exo compared to M0-exo. GO and KEGG enrichment analysis revealed that the downstream targets of DE-miRNAs were mainly involved in the differentiation and migration of multiple cells.ConclusionsIn summary, we have indicated for the first time that M2-exo can promote osteogenic differentiation of human PDLSCs, and have revealed the functions and pathways involved in the DE-miRNAs of M0-exo and M2-exo and their downstream targets.

High formability Mg-Zn-Gd wire facilitates ACL reconstruction via its swift degradation to accelerate intra-tunnel endochondral ossification

After reconstructing the anterior cruciate ligament (ACL), unsatisfactory bone tendon interface healing may often induce tunnel enlargement at the early healing stage. With good biological features and high formability, Magnesium-Zinc-Gadolinium (ZG21) wires are developed to bunch the tendon graft for matching the bone tunnel during transplantation. Microstructure, tensile strength, degradation, and cytotoxicity of ZG21 wire are evaluated. The rabbit model is used for assessing the biological effects of ZG21 wire by Micro-CT, histology, and mechanical test. The SEM/EDS, immunochemistry, and in vitro assessments are performed to investigate the underlying mechanism. Material tests demonstrate the high formability of ZG21 wire as surgical suture. Micro-CT shows ZG21 wire degradation accelerates tunnel bone formation, and histologically with earlier and more fibrocartilage regeneration at the healing interface. The mechanical test shows higher ultimate load in the ZG21 group. The SEM/EDS presents ZG21 wire degradation triggered calcium phosphate (Ca-P) deposition. IHC results demonstrate upregulation of Wnt3a, BMP2, and VEGF at the early phase and TGFβ3 and Type II collagen at the late phase of healing. In vitro tests also confirmed the Ca-P in the metal extract could elevate the expression of Wnt3a, β catenin, ocn and opn to stimulate osteogenesis. Ex vivo tests of clinical samples indicated suturing with ZG21 wire did not weaken the ultimate loading of human tendon tissue. In conclusion, the ZG21 wire is feasible for tendon graft bunching. Its degradation products accelerated intra-tunnel endochondral ossification at the early healing stage and therefore enhanced bone-tendon interface healing in ACL reconstruction.

MicroRNA-142–3p regulates osteogenic differentiation of human periodontal ligament stem cells via mediating SGK1

ObjectiveHuman periodontal ligament stem cells (hPDLSCs) refer to one kind of somatic stem cells that are capable of differentiating into multiple cell kinds and undergoing robust clonal self-renewal. This work was unearthed to elucidate the possible molecular mechanism of miR-142–3p in mediating osteogenic differentiation of hPDLSCs by targeting SGK1. MethodsThe hPDLSCs were isolated, cultured, and identified. hPDLSCs were identified by immunofluorescence staining and multiple differentiation ability detection. Cell proliferation ability was assessed by CCK-8 assay. hPDLSCs were induced using osteogenic differentiation medium. ALP activity was detected by alkaline phosphatase (ALP) staining and ALP activity assay, and mineralized nodule formation was determined by alizarin red staining. The expression levels of osteogenic differentiation marker proteins ALP, RUNX2, and OCN were measured by RT-qPCR. miR-142–3p candidate targets were obtained through bioinformatics analysis. The relationship between miR-142–3p and SKG1 was verified. ResultsmiR-142–3p in hPDLSCs after osteogenic induction was down-regulated. Elevated miR-142–3p restricted hPDLSCs proliferation, and diminished ALP activity and mineralized nodule formation, as well as the expression of ALP, RUNX2, and OCN, while miR-142–3p inhibition led to inverse results. miR-142-3p inhibited SKG1 expression. SKG1 overexpression promoted hPDLSC proliferation and osteogenic differentiation, and reversed the inhibitory function of miR-142–3p on hPDLSCs. ConclusionThis study highlights that miR-142–3p represses osteogenic differentiation of hPDLSCs by reducing SGK1 expression.

Biomimetic Hydroxyapatite Composite Coatings with a Variable Morphology Mediated by Silk Fibroin and Its Derived Peptides Enhance the Bioactivity on Titanium.

Various modifications performed on titanium alloy surfaces are shown to improve osteointegration and promote the long-term success of implants. In this work, a bioactive nanostructured hydroxyapatite (HA) composite coating with a variable morphology mediated by silk fibroin (SF) and its derived peptides (Cs) was prepared. Numerous experimental techniques were used to characterize the constructed coatings in terms of morphology, roughness, hydrophilicity, protein adsorption, in vitro biomineralization, and adhesion strength. The mixed protein layer with different contents of SF and Cs exhibited different secondary structures at different temperatures, effectively mediating the electrodeposited HA layer with different characteristics and finally forming proteins/HA composite coatings with versatile morphologies. The addition of Cs significantly improved the hydrophilicity and protein adsorption capacity of the composite coatings, while the electrodeposition of the HA layer effectively enhanced the adhesion between the composite coatings and Ti surface. In the in vitro mineralization experiments, all the composite coatings exhibited excellent apatite formation ability. Moreover, the composite coatings showed excellent cell growth and proliferation activity. Osteogenic induction experiments revealed that the coating could significantly increase the expression of specific osteogenic markers, including ALP, Col-I, Runx-2, and OCN. Overall, the proposed modification of the Ti implant surface by protein/HA coatings had good potential for clinical applications in enhancing bone induction and osteogenic activity of implants.

Surface modification of nanofibrous polyethersulfone scaffolds with fluorapatite nanoparticles toward improved stem cell behavior and osteogenic activity in vitro

Nanofibrous polyethersulfone (PESf) scaffolds were fabricated by the electrospinning method. The fabricated scaffolds were surface-modified by fluorapatite nanoparticles (FAn) synthesized using Reline deep eutectic solvent (DES). The effect of surface modification on microstructure, wettability, porosity, biodegradation, and absorption of the scaffolds was investigated. Results confirmed the formation of nanofibers with narrow width between 250 and 600 nm and length of a few tens of microns which was roughened by nanoscale spherical FA particles having a particle size in the range of 40–60 nm. Surface modification of PES by FA nanoparticles led to a significant increase in hydrophilicity, roughness, porosity, and biodegradation which in turn resulted in improvement of viability, growth, proliferation, and differentiation of human bone marrow mesenchymal stem cells (hBMMSCs) assessed by MTT, calcium content (biomineralization), alkaline phosphatase (ALP) activity, alizarin red, and acridine orange staining tests. Gene expression test represented a significant increase in expression of ALP, OCN, RUNX-2, ONN, and COL-I in differentiated stem cells which was attributed to the changes in surface topography and releasing bioactive calcium and fluoride ions made by the inclusion of FA nanoparticles. In conclusion, the nanoscale FA particles in the nanofibrous PES scaffolds provided multiple avenues to engineer PES by modification of surface characteristics for favorable cell-surface interactions, biocalcification, and osteogenesis.

Melatonin promoted osteogenesis of human periodontal ligament cells by regulating mitochondrial functions through the translocase of the outer mitochondrial membrane 20.

Melatonin plays an important role in various beneficial functions, including promoting differentiation. However, effects on osteogenic differentiation, especially in human periodontal cells (hPDLCs), still remain inconclusive. Mitochondria are highly dynamic organelles that play an important role in various biological processes in cells, including energy metabolism and oxidative stress reaction. Furthermore, the translocase of the outer mitochondrial membrane 20 (TOM20) is responsible for recognizing and transporting precursor proteins. Thus, the objective of this study was to evaluate the functionality of melatonin on osteogenesis in human periodontal cells and to explore the involved mechanism of mitochondria. The hPDLCs were extracted and identified by flow cytometry and multilineage differentiation. We divided hPDLCs into control group, osteogenic induction group, and osteogenesis with melatonin treatment group (100, 10, and 1μM). Then we used a specific siRNA to achieve interference of TOM20. Alizarin red and Alkaline phosphatase staining and activity assays were performed to evaluate osteogenic differentiation. Osteogenesis-related genes and proteins were measured by qPCR and western blot. Mitochondrial functions were tested using ATP, NAD+/NADH, JC-1, and Seahorse Mito Stress Test kits. Finally, TOM20 and mitochondrial dynamics-related molecules expression were also assessed by qPCR and western blot. Our results showed that melatonin-treated hPDLCs had higher calcification and ALP activity as well as upregulated OCN and Runx2 expression at mRNA and protein levels, which was the most obvious in 1μM melatonin-treated group. Meanwhile, melatonin supplement elevated intracellular ATP production and mitochondrial membrane potential by increasing mitochondrial oxidative metabolism, hence causing a lower NAD+ /NADH ratio. In addition, we also found that melatonin treatment raised TOM20 level and osteogenesis and mitochondrial functions were both suppressed after knocking down TOM20. We found that melatonin promoted osteogenesis of hPDLCs and 1μM melatonin had the most remarkable effect. Melatonin treatment can reinforce mitochondrial functions by upregulating TOM20.

Mir-184 Regulates Bone Marrow Stromal Cells in Osteoporosis Rats via Targeting Special AT-Rich Sequence-Binding Protein 2 (Satb2)

Bone marrow stromal cells (BMSCs) participates in osteoporosis (OP) development and their differentiation can affect OP progression. miR-184 is involved in several diseases. However, miR-184’s role in BMSCs in osteoporosis is unclear. Rat BMSCs were isolated and assigned into control group, Mir-184 inhibitor group and Mir-184 group followed by analysis of Mir-184, Runx2 and OCN expression by Real time PCR, ALP activity and calcified nodules formation by Alizarin red staining. SD rats were separated into sham operation group, OP group and Mir-184 inhibitor group and bone density was assessed by micro-CT. Mir-184 inhibitor transfection significantly down-regulated Mir-184, increased expression of Runx2, OCN and Stab2 and promoted ALP activity (P<0.05), which were all reversed by Mir-184 mimic. Satb2 was targeted by Mir-184. Transfection of Mir-184 inhibitor and shSATB2 lentivirus decreased Runx2 and OCN expression and reduced calcified nodules formation. Mir-184 down-regulation in OP rats promoted Runx2 and OCN expression and increased bone density. In conclusion, Mir-184 expression is increased in OP rat BMSCs and its down-regulation can target Satb2, which promote osteogenic differentiation and increase calcified nodule formation and ameliorate OP disease.

Effects of Human Deciduous Dental Pulp-Derived Mesenchymal Stem Cell-Derived Conditioned Medium on the Metabolism of HUVECs, Osteoblasts, and BMSCs.

In this study, we assessed the effects of human deciduous dental pulp-derived mesenchymal stem cell-derived conditioned medium (SHED-CM) on the properties of various cell types. The effects of vascular endothelial growth factor (VEGF) in SHED-CM on the luminal architecture, proliferative ability, and angiogenic potential of human umbilical vein endothelial cells (HUVECs) were determined. We also investigated the effects of SHED-CM on the proliferation of human-bone-marrow mesenchymal stem cells (hBMSCs) and mouse calvarial osteoblastic cells (MC3T3-E1) as well as the expression of ALP, OCN, and RUNX2. The protein levels of ALP were examined using Western blot analysis. VEGF blockade in SHED-CM suppressed the proliferative ability and angiogenic potential of HUVECs, indicating that VEGF in SHED-CM contributes to angiogenesis. The culturing of hBMSCs and MC3T3-E1 cells with SHED-CM accelerated cell growth and enhanced mRNA expression of bone differentiation markers. The addition of SHED-CM enhanced ALP protein expression in hBMSCs and MT3T3-E1 cells compared with that of the 0% FBS group. Furthermore, SHED-CM promoted the metabolism of HUVECs, MC3T3-E1 cells, and hBMSCs. These findings indicate the potential benefits of SHED-CM in bone tissue regeneration.

A comparative study of the morphology and molecular biology between the Schneiderian membrane and palatine mucoperiosteum

Schneiderian membrane is an indispensable structure for osteogenesis under the sinus floor space after maxillary sinus floor elevation.Therefore,this study aimed to compare the Schneiderian membrane and palatine mucoperiosteum in various aspects to explore whether the Schneiderian membrane has a periosteal layer and osteogenic ability. Schneiderian membrane and palatine mucoperiosteum specimens were collected and stained with HE, Masson, and Sirius red. Immunofluorescence staining was used to observe the expression and localization of mesenchymal stem cells (MSCs). Then MSCs from two tissues were isolated,cultured, and identified. The expression of osteogenic markers OCN, RUNX2, and BMP2 ware detected by Western blotting and quantitative PCR after osteogenic differentiation.The morphological observations revealed both the Schneiderian membrane and palatine mucoperiosteum were composed of three layers.Immunofluorescence staining showed that the inner bone surface layer of the Schneiderian membrane was rich in MSCs, which was similar to the cambium layer of the palatine mucoperiosteum.In addition, MSCs from two tissues showed similar morphological phenotype. After further osteogenic induction of the two groups, the expression of BMP2, RUNX2, and OCN were significantly increased. This study provide a novel insight into that Schneiderian membrane is a mucoperiosteal membrane rich of MSCs, containing a periosteal layer and osteogenic ability similar to mucoperiosteum.

Irisin promotes fracture healing by improving osteogenesis and angiogenesis.

BackgroundOsteogenesis and angiogenesis are important for bone fracture healing. Irisin is a muscle-derived monokine that is associated with bone formation.MethodsTo demonstrate the effect of irisin on bone fracture healing, closed mid-diaphyseal femur fractures were produced in 8-week-old C57BL/6 mice. Irisin was administrated intraperitoneally every other day after surgery, fracture healing was assessed by using X-rays. Bone morphometry of the fracture callus were assessed by using micro-computed tomography. Femurs of mice from each group were assessed by the three-point bending testing. Effect of irisin on osteogenic differentiation in mesenchymal stem cells in vitro was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), alkaline phosphatase staining and alizarin red staining. Angiogenesis of human umbilical vein endothelial cells (HUVECs) were evaluated by qRT-PCR, migration tests, and tube formation assays.ResultsIncreased callus formation, mineralization and tougher fracture healing were observed in the irisin-treated group than in the control group, indicating the better fracture callus healing due to Irisin treatment. The vessel surface and vessel volume fraction of the callus also increased in the irisin-treated group. The expression of BMP2, CD31, and VEGF in callus were enhanced in the irisin-treated group. In mouse bone mesenchymal stem cells, irisin promoted ALP expression and mineralization, and increased the expression of osteogenic genes, including OSX, Runx2, OPG, ALP, OCN and BMP2. Irisin also promoted HUVEC migration and tube formation. Expression of angiogenic genes, including ANGPT1, ANGPT2, VEGFb, CD31, FGF2, and PDGFRB in HUVECs were increased by irisin.ConclusionAll the results indicate irisin can promote fracture healing through osteogenesis and angiogenesis. These findings help in the understanding of muscle–bone interactions during fracture healing.The Translational Potential of this ArticleIrisin was one of the most important monokine secreted by skeletal muscle. Studies have found that irisin have anabolic effect one bone remodeling through affecting osteocyte and osteoblast. Based on our study, irisin could promote bone fracture healing by increasing bone mass and vascularization, which provide a potential usage of irisin to promote fracture healing and improve clinical outcomes.

Experimental study of resveratrol-solid lipid nanoparticles in promotion of osteogenic differentiation of bone marrow mesenchymal stem cells

To investigate the effect of solid lipid nanoparticles (SLNs) on enhancing the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro by resveratrol (Res), and provide a method for the treatment of bone homeostasis disorders. Res-SLNs were prepared by high-temperature emulsification and low-temperature solidification method, and then the 2nd-3rd generation BMSCs from Sprague Dawley rat were co-cultured with different concentrations (0, 0.1, 1, 5, 10, 20 μmol/L) of Res and Res-SLNs. The effects of Res and Res-SLNs on the cell viability of BMSCs were detected by cell counting kit 8 (CCK-8) and live/dead cell staining; the effects of Res and Res-SLNs on the osteogenic differentiation of BMSCs were detected by alkaline phosphatase (ALP) staining and alizarin red S (ARS) staining after osteogenic differentiation induction, and the optimal concentration of Res-SLNs for gene detection was determined. Anti-osteocalcin (OCN) immunofluorescence staining and real-time fluorescent quantitative PCR (RT-qPCR) were used to detect the effect of Res and Res-SLNs on osteoblast-related genes (ALP and OCN) of BMSCs. Live/dead cell staining showed that there was no significant difference in the number of dead cells between Res and Res-SLNs groups; CCK-8 detection showed that the activity of BMSCs in Res group was significantly reduced at the concentration of 20 μmol/L (P<0.05), while Res-SLNs activity was not affected by Res concentration (P>0.05). After osteogenic differentiation, the staining intensity of ALP and ARS in both groups was dose-dependent. The percentage of ALP positive staining area and the percentage of mineralized nodule area in Res group and Res-SLNs group reached the maximum at the concentrations of 10 μmol/L and 1 μmol/L, respectively (P<0.05), and then decreased gradually; the most effective concentration of Res-SLNs was 1 μmol/L. The expression of OCN and the relative expression of ALP and OCN mRNA in Res-SLNs group were significantly higher than those in Res group (P<0.05). Encapsulation of SLNs can improve the effect of Res on promoting osteogenesis, and achieve the best effect of osteogenic differentiation of BMSCs at a lower concentration, which is expected to be used in the treatment of bone homeostasis imbalance diseases.

Low-level laser therapy with different irradiation methods modulated the response of bone marrow mesenchymal stem cells in vitro.

Low-level laser therapy (LLLT) also known as photobiomodulation is a treatment to change cellular biological activity. The exact effects of LLLT remain unclear due to the different irradiation protocols. The purpose of this study was to investigate the effects of LLLT by three different irradiation methods on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro. BMSCs were inoculated in 24-well plates and then irradiated or not (control) with a laser using three different irradiation methods. The irradiation methods were spot irradiation, covering irradiation, and scanning irradiation according to different spot areas (0.07 cm2 or 1.96 cm2) and irradiation areas (0.35 cm2 or 1.96 cm2), respectively. The laser was applied three times at energy densities of 4J/cm2. The cell proliferation by CCK-8. ALP activity assay, alizarin red, and quantitative real-time polymerase chain reaction (RT-PCR) were performed to assess osteogenic differentiation and mineralization. Increases in cell proliferation was obvious following irradiation, especially for covering irradiation. The ALP activity was significantly increased in irradiated groups compared with non-irradiated control. The level of mineralization was obviously improved following irradiation, particularly for covering irradiation. RT-PCR detected significantly higher expression of ALP, OPN, OCN, and RUNX-2 in the group covering than in the others, and control is the lowest. The presented results indicate that the biostimulative effects of LLLT on BMSCs was influenced by t he irradiation method, and the covering irradiation is more favorable method to promote the proliferation and osteogenic differentiation of BMSCs.

Osteogenic effect of electromagnetic fields on stem cells derived from rat bone marrow cultured in osteogenic medium versus conditioned medium in vitro.

This study assessed possible osteogenic differentiation caused by electromagnetic fields (EMF) on rat bone-marrow-derived stem cells (rBMSCs) cultured in osteogenic medium (OM) or in human adipose-stem cell-conditioned medium (hADSC-CM). The rBMSCs were divided into negative and positive control groups, cultured in α-MEM plus 10% FBS or OMrespectively. CM and CM + EMF groups, culturedcells in hADSCs-CM or exposed to EMF (50Hz, 1mT) for 30min/day plus hADSCs-CM, respectively. Cells from the OM + EMF were simultaneously cultured in OM and exposed to EMF. Osteogenesis was investigated through alkaline phosphatase activity, alizarin red staining and real-time PCR. A meaningfully higher level of ALP activity was observed in the OM + EMF group compared to the other groups. There was a considerable increase in Runx2 expression in the CM + EMF group compared to the positive control and CM groups and a significant increase in Runx2 expression in the OM + EMF in comparison with all other groups after 21days. Runx2 expression increased significantly in the CM, CM + EMF and positive control groups on day 21 compared to the same groups on day 14. From days 14-21, Ocn expression increased in the CM and CM + EMF groups, but both groups showed a significant decrease compared to the positive controls. CM and EMF had no effect on Ocn expression. On day 21, Ocn expression was significantly higher in the OM + EMF group than in the positive control group. The synergistic effect of EMF and OM increased the expression of Runx2 and Ocn in rBMSCs.

Effect and mechanism of a novel Mg-Nd-Gd-Sr alloy on osteogenic differentiation of bone marrow mesenchymal stem cells.

We investigated the effect and mechanism of a novel Mg-3Nd-1Gd-0.3Sr-0.2Zn-0.4Zr (abbreviated to Mg-Nd-Gd-Sr) alloy on the osteogenic differentiation of bone marrow mesenchymal stem cells extracted from Sprague-Dawley rats. Cultured cells were divided into five groups: a control group cultured in osteogenic induction medium alone without Mg-Nd-Gd-Sr alloy extract, and four experimental groups cultured in the same medium with 25%, 50%, 75%, and 100% Mg-Nd-Gd-Sr alloy extracts, respectively. After 14 days of culture, ALP activity was determined and expressions of osteogenesis-related factors Runx2, OCN, and OPN at the mRNA level and Runx2, OCN, and OPN at the protein level were detected by RT-PCR and western blot, respectively. After 21days of culture, mineralized nodules were detected by alizarin red staining. The results showed that bone marrow mesenchymal stem cells from Sprague-Dawley rats were successfully isolated in vitro using the whole bone marrow adherence method. Flow cytometry revealed that the cells expressed high levels of CD44 and CD90, but low levels of CD31 and CD45. Alizarin red staining indicated the formation of mineralized nodules in all five groups. Compared with the control group, the number of mineralized nodules was increased significantly in the four experimental groups (p < 0.05). The ALP activity in each group was significantly higher on day 14 than on day 7, and was significantly higher in the four experimental groups compared with the control group (p < 0.05). Moreover, the ALP activity was highest when the concentration of Mg-Nd-Gd-Sr alloy extract was 75% (p < 0.05). RT-PCR results showed that, compared with the control group, the mRNA expression of Runx2, OPN, and OCN was significantly higher in the four experimental groups (p < 0.05), and the highest mRNA expression of Runx2, OPN, and OCN was observed in the 75% experimental group (p < 0.05). Western blotting showed that Mg-Nd-Gd-Sr alloy extract significantly increased the protein expression of Runx2, OCN, and OPN compared with the control group (p < 0.05). Our data indicate that the novel Mg-Nd-Gd-Sr alloy can promotes the osteogenic differentiation of bone marrow mesenchymal stem cells isolated from Sprague-Dawley rats. During this process, there is an increase in the expressions of Runx2, OPN, and OCN mRNAs and Runx2, OCN, and OPN proteins.

Beclin1- and Atg13-dependent autophagy activation and morroniside have synergistic effect on osteoblastogenesis.

Morroniside is known to improve osteoporosis by promoting osteoblastogenesis. The activation of PI3K/Akt/mTOR signaling is a significant mechanism in morroniside-promoted osteoblastogenesis. It is well known that protective autophagy is an important factor in osteoblastogenesis. However, the activation of mTOR signaling can inhibit autophagy. This study aimed to investigate the relationship between mTOR signaling and autophagy in morroniside-regulated osteoblastogenesis. In this study, we investigated the effect of morroniside on the autophagic activity (LC3 conversion rate, LC3-puncta formation, and autophagosome number) of differentiated osteoblast precursors (MC3T3-E1 cells). Then, we identified the roles of mTOR knockdown in morroniside-regulated alterations of autophagy and osteogenic parameters in MC3T3-E1 cells. Next, mTOR knockdown and overexpression were used to observe the roles of mTOR in morroniside-regulated alterations of autophagic molecules (Atg7, Atg13, and Beclin1). Subsequently, the additional value of the above autophagic molecules on morroniside-regulated osteogenic parameters in MC3T3-E1 cells was analyzed based on lentiviral transduction. Finally, combined with morroniside and TAT-Beclin1, the roles of Beclin1 upregulation in the in vivo effects of morroniside was investigated. Our experimental data showed that morroniside promoted both the mTOR activity and autophagy in MC3T3-E1 cells. Morroniside-upregulated autophagic activity and Atg13 or Beclin1 protein level in MC3T3-E1 cells were enhanced by mTOR knockdown. Furthermore, Morroniside-upregulated Atg13 and Beclin1 expression was reversed by mTOR overexpression. Importantly, autophagy upregulation with overexpression of the autophagic gene, Atg13 or BECN1 (gene form of Beclin1), significantly promoted osteoblastogenesis regulated by morroniside. The promotional effect of morroniside on bone microarchitecture, bone mass, and bone parameters (including trabecular bone area and OCN expression in trabecular bone) in ovariectomized (OVX) mice was enhanced by TAT-Beclin1 administration. In conclusion, the autophagy-enhancing drugs related to Beclin1 or Atg13 may be an effective adjuvant therapy in the treatment of osteoporosis with morroniside.

6-Bromoindirubin-3'-Oxime Regulates Colony Formation, Apoptosis, and Odonto/Osteogenic Differentiation in Human Dental Pulp Stem Cells.

6-bromoindirubin-3′-oxime (BIO) is a candidate small molecule that effectively modulates Wnt signalling owing to its stable property. The present study investigated the influence of BIO on the odonto/osteogenic differentiation of human dental pulp stem cells (hDPSCs). hDPSCs were treated with 200, 400, or 800 nM BIO, and the effects on hDPSC responses and osteogenic differentiation were assessed. BIO-mediated Wnt activation was confirmed by β-catenin nuclear translocation detected by immunofluorescence staining. BIO attenuated colony formation and cell migration determined by in vitro wound-healing assay. BIO increased early apoptotic cell population evaluated using flow cytometry. For osteogenic induction, BIO promoted alkaline phosphatase (ALP) activity and mineralisation in a dose-dependent manner. ALP, RUNX2, OCN, OSX, ANKH, DMP1, and DSPP mRNA expression were significantly upregulated. The OPG/RANKL expression ratio was also increased. Further, BIO attenuated adipogenic differentiation as demonstrated by decreased lipid accumulation and adipogenic-related gene expression. Bioinformatic analysis of RNA sequencing data from the BIO-treated hDPSCs revealed that BIO modulated pathways related to autophagy and actin cytoskeleton regulation. These findings demonstrated that BIO treatment promoted hDPSC osteogenic differentiation. Therefore, this small molecule is a strong candidate as a bioactive molecule to enhance dentin repair.

Effect of Alumina Particles on the Osteogenic Ability of Osteoblasts.

Biomaterials are used as implants for bone and dental disabilities. However, wear particles from the implants cause osteolysis following total joint arthroplasty (TJA). Ceramic implants are considered safe and elicit a minimal response to cause periprosthetic osteolysis. However, few reports have highlighted the adverse effect of ceramic particles such as alumina (Al2O3) on various cell types. Hence, we aimed to investigate the effect of Al2O3 particles on osteoprogenitors. A comparative treatment of Al2O3, Ti, and UHMWPE particles to osteoprogenitors at a similar concentration of 200 μg/mL showed that only Al2O3 particles were able to suppress the early and late differentiation markers of osteoprogenitors, including collagen synthesis, alkaline phosphatase (ALP) activity and mRNA expression of Runx2, OSX, Col1α, and OCN. Al2O3 particles even induced inflammation and activated the NFkB signaling pathway in osteoprogenitors. Moreover, bone-forming signals such as the WNT/β-catenin signaling pathway were inhibited by the Al2O3 particles. Al2O3 particles were found to induce the mRNA expression of WNT/β-catenin signaling antagonists such as DKK2, WIF, and sFRP1 several times in osteoprogenitors. Taken together, this study highlights a mechanistic view of the effect of Al2O3 particles on osteoprogenitors and suggests therapeutic targets such as NFĸB and WNT signaling pathways for ceramic particle-induced osteolysis.

O096 Does boron bioactive glass have a role in bone regeneration?

Abstract Introduction Bioactive glasses (BGs) provide an alternative to allogenic bone grafts. Various ions can be incorporated into the BGs structure to further influence regenerative or antimicrobial properties. Boron has recently been purposed as element that can be incorporated into BGs to promote desirable cellular regenerative response. This study aims to systematically review literature to determine the level of evidence that boron bioactive glasses (BBGs) and boron can promote desirable bone regenerative responses both in vitro and in vivo. Methods A systematic review of Web of Science was conducted in accordance with PRISMA guidelines. Data collected included bone cell behavioural analysis in response to boron or BBG compared to controls. Material properties of BBG (mechanical properties, degradation rate) compared to BG were compared. Results A total of 105 articles were included; 98 regarding BBG, and 12 articles studying the effect of boron. A higher biodegradation rate was observed in BBG compared with silicate-based BGs. BBGs have a lower compressive strength and increased fragility, as the percentage of B203 increases. 16 articles demonstrated a positive correlation with BBG and expression of OCN, VEGF, osteopontin, and RUNX2. Overall, metabolic activity of cells up to 14 days was lower in comparison to media and silicate BG. Conclusion BBGs have gained increased attention in the literature but the lack of heterogeneity and paucity of data does not provide convincing evidence of the effect of boron on bone regeneration and indicates the need for further quantitative research with a standardised approach to assessment. Take-home message Bioactive glasses can promote regeneration of bone, however the lack of heterogeneity and paucity of data regarding boron bioactive glass indicate the need for further quantitative research and standardised approach to assessment.

Metformin activates Wnt/β-catenin for the treatment of diabetic osteoporosis

BackgroundWith the deepening of social aging, the incidence rate of osteoporosis and diabetes continues to rise. More and more clinical studies show that diabetes is highly correlated with osteoporosis. Diabetes osteoporosis is considered as a metabolic bone disease of diabetes patients. This study aims to explore the role and mechanism of metformin (Met) in diabetic osteoporosis.MethodsMouse MC3T3-E1 cells were treated with Met (0.5 mM) and exposed to high glucose (HG, 35 mM). The cells were cultured in an osteogenic medium for osteogenic differentiation, and the cell proliferation ability was determined using Cell Counting Kit-8; Alkaline phosphatase (ALP) activity detection and alizarin red staining were utilized to evaluate the effect of Met on MC3T3-E1 osteogenic differentiation. Western blot was used to detect the expressions of osteogenesis-related proteins (Runx2 and OCN) as well as Wnt/β-catenin signaling pathway-related proteins in MC3T3-E1 cells.ResultsHG inhibited proliferation and calcification of MC3T3-E1 cells, down-regulated ALP activity, and the expression of Runx2 and OCN in MC3T3-E1 cells. Meanwhile, the activity of the Wnt/β-catenin signaling pathway was inhibited. Met treatment was found to significantly stimulate the proliferation and calcification of MC3T3-E1 cells under HG conditions, as well as increase the ALP activity and the protein expression level of Runx2 and OCN in the cells. As a result, osteogenic differentiation was promoted and osteoporosis was alleviated. Apart from this, Met also increased the protein expression level of Wnt1, β-catenin, and C-myc to activate the Wnt/β-catenin signaling pathway.ConclusionMet can stimulate the proliferation and osteogenic differentiation of MC3T3-E1 cells under HG conditions. Met may also treat diabetic osteoporosis through Wnt/β-catenin activation.

Silicocarnotite: Novel Silicate Bioceramic With Osteogenic Property for Repairing Rat Cranial Critical-Sized Bone Defects

Critical-sized bone defects are an intractable orthopedic disease which often fails to regenerate spontaneously and requires additional intervention. Current therapies, including autografts and allografts, are not always satisfactory. Herein, the novel calcium phosphate bioceramic-containing silicon (CPS) with a carnotite structure was synthesized. In the present study, CPS was prepared for investigating the biocompatibility and bioactivity in vitro and in vivo in comparison to hydroxyapatite (HA). Our results showed that CPS bioceramics had favorable biocompatibility and rBMSCs could adhere on the surface well in vitro. Moreover, CPS could promote osteogenic differentiation of rBMSCs and the expression of osteogenic differentiation marker genes, including ALP, Runx-2, BSP, OCN, and OPN. In vivo, the results of micro-CT, histomorphometry, and histology analyses showed that CPS significantly enhanced critical-sized calvarial defects healing compared with HA. Overall, the present study demonstrated that CPS bioceramics had satisfactory bioactivities and osteogenic capacities, which could be a potential option for reconstructing critical-sized bone defects.